Montelukast Inhibits Platelet Activation Induced by Plasma From COVID-19 Patients.

Leukotrienes are important pro-inflammatory lipid mediators derived from the arachidonic acid metabolism. In particular, cysteinyl leukotrienes, namely LTC4, LTD4, and LTE4 are involved in many of the principal features of asthma, while more recently they have also been implicated in cardiovascular diseases. COVID-19 is characterized by an overwhelming state of inflammation, sometimes resulting in an acute respiratory distress syndrome. Furthermore, severe COVID-19 patients present an endothelial cell damage characterized by a hyperinflammatory/procoagulant state and a widespread thrombotic disease. Leukotriene receptor antagonists, such as montelukast, have long been proven to have an efficacy in asthma, while more recently they have been suggested to have a protective role also in cardiovascular diseases. As elevated levels of LTE4 have been detected in bronchoalveolar lavage of COVID-19 patients, and montelukast, in addition to its anti-inflammatory properties, has been suggested to have a protective role in cardiovascular diseases, we decided to investigate whether this drug could also affect the platelet activation characteristic of COVID-19 syndrome. In this contribution, we demonstrate that montelukast inhibits platelet activation induced by plasma from COVID-19 patients by preventing the surface expression of tissue factor (TF) and P-selectin, reducing the formation of circulating monocyte- and granulocyte-platelet aggregates, and, finally, in completely inhibiting the release of TFpos-circulating microvesicles. These data suggest the repurposing of montelukast as a possible auxiliary treatment for COVID-19 syndrome.

Effects of non-steroidal anti-inflammatory drugs and other eicosanoid pathway modifiers on antiviral and allergic responses: EAACI task force on eicosanoids consensus report in times of COVID-19.

Non-steroidal anti-inflammatory drugs (NSAIDs) and other eicosanoid pathway modifiers are among the most ubiquitously used medications in the general population. Their broad anti-inflammatory, antipyretic, and analgesic effects are applied against symptoms of respiratory infections, including SARS-CoV-2, as well as in other acute and chronic inflammatory diseases that often coexist with allergy and asthma. However, the current pandemic of COVID-19 also revealed the gaps in our understanding of their mechanism of action, selectivity, and interactions not only during viral infections and inflammation, but also in asthma exacerbations, uncontrolled allergic inflammation, and NSAIDs-exacerbated respiratory disease (NERD). In this context, the consensus report summarizes currently available knowledge, novel discoveries, and controversies regarding the use of NSAIDs in COVID-19, and the role of NSAIDs in asthma and viral asthma exacerbations. We also describe here novel mechanisms of action of leukotriene receptor antagonists (LTRAs), outline how to predict responses to LTRA therapy and discuss a potential role of LTRA therapy in COVID-19 treatment. Moreover, we discuss interactions of novel T2 biologicals and other eicosanoid pathway modifiers on the horizon, such as prostaglandin D2 antagonists and cannabinoids, with eicosanoid pathways, in context of viral infections and exacerbations of asthma and allergic diseases. Finally, we identify and summarize the major knowledge gaps and unmet needs in current eicosanoid research.

Current perspective on eicosanoids in asthma and allergic diseases: EAACI Task Force consensus report, part I.

Eicosanoids are biologically active lipid mediators, comprising prostaglandins, leukotrienes, thromboxanes, and lipoxins, involved in several pathophysiological processes relevant to asthma, allergies, and allied diseases. Prostaglandins and leukotrienes are the most studied eicosanoids and established inducers of airway pathophysiology including bronchoconstriction and airway inflammation. Drugs inhibiting the synthesis of lipid mediators or their effects, such as leukotriene synthesis inhibitors, leukotriene receptors antagonists, and more recently prostaglandin D2 receptor antagonists, have been shown to modulate features of asthma and allergic diseases. This review, produced by an European Academy of Allergy and Clinical Immunology (EAACI) task force, highlights our current understanding of eicosanoid biology and its role in mediating human pathology, with a focus on new findings relevant for clinical practice, development of novel therapeutics, and future research opportunities.

Montelukast Use Decreases Cardiovascular Events in Asthmatics.

Cysteinyl leukotrienes are proinflammatory mediators with a clinically established role in asthma and a human genetic and preclinical role in cardiovascular pathology. Given that cardiovascular disease has a critical inflammatory component, the aim of this work was to conduct an observational study to verify whether the use of a cysteinyl leukotriene antagonist, namely, montelukast, may protect asthmatic patients from a major cardiovascular event and, therefore, represent an innovative adjunct therapy to target an inflammatory component in cardiovascular disease. We performed an observational retrospective 3-year study on eight hundred adult asthmatic patients 18 years or older in Albania, equally distributed into two cohorts, exposed or nonexposed to montelukast usage, matched by age and gender according to information reported in the data collection. Patients with a previous history of myocardial infarction or ischemic stroke were excluded. In summary, 37 (4.6%) of the asthmatic patients, 32 nonexposed, and five exposed to montelukast suffered a major cardiovascular event during the 3-year observation period. All the cardiovascular events, in either group, occurred among patients with an increased cardiovascular risk. Our analyses demonstrate that, independent from gender, exposure to montelukast remained a significant protective factor for incident ischemic events (78% or 76% risk reduction depending on type of analysis). The event-free Kaplan-Meier survival curves confirmed the lower cardiovascular event incidence in patients exposed to montelukast. Our data suggest that there is a potential preventative role of montelukast for incident cardiac ischemic events in the older asthmatic population, indicating a comorbidity benefit of montelukast usage in asthmatics by targeting cysteinyl leukotriene-driven cardiac disease inflammation.

Arachidonic Acid and Docosahexaenoic Acid Metabolites in the Airways of Adults With Cystic Fibrosis: Effect of Docosahexaenoic Acid Supplementation.

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by genetic mutations in CF transmembrane conductance regulator protein. Several reports have indicated the presence of specific fatty acid alterations in CF patients, most notably decreased levels of plasmatic and tissue docosahexaenoic acid (DHA), the precursor of specialized pro-resolving mediators. We hypothesized that DHA supplementation could restore the production of DHA-derived products and possibly contribute to a better control of the chronic pulmonary inflammation observed in CF subjects. Sputum samples from 15 CF and 10 chronic obstructive pulmonary disease (COPD) subjects were collected and analyzed by LC/MS/MS, and blood fatty acid were profiled by gas chromatography upon lipid extraction and transmethylation. Interestingly, CF subjects showed increased concentrations of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and 15-hydroxyeicosatetraenoic acid (15-HETE), when compared with COPD patients, whereas the concentrations of DHA metabolites did not differ between the two groups. After DHA supplementation, not only DHA/arachidonic acid (AA) ratio and highly unsaturated fatty acid index were significantly increased in the subjects completing the study (p < 0.05) but also a reduction in LTB4 and 15-HETE was observed, together with a tendency for a decrease in PGE2, and an increase in 17-hydroxy-docosahexaenoic acid (17OH-DHA) levels. At the end of the washout period, LTB4, PGE2, 15-HETE, and 17OH-DHA showed a trend to return to baseline values. In addition, 15-HETE/17OH-DHA ratio in the same sample significantly decreased after DHA supplementation (p < 0.01) when compared with baseline. In conclusion, our results show here that in CF patients, an impairment in fatty acid metabolism, characterized by increased AA-derived metabolites and decreased DHA-derived metabolites, could be partially corrected by DHA supplementation.

Discovery of the First in Vivo Active Inhibitors of the Soluble Epoxide Hydrolase Phosphatase Domain.

The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities that are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity, located in the N-terminal phosphatase domain of sEH (sEH-P). Herein we report the discovery and optimization of the first inhibitor of human and rat sEH-P that is applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.

Two-pronged approach to anti-inflammatory therapy through the modulation of the arachidonic acid cascade.

Chronic inflammation and pain is a major global health problem, and nonsteroidal anti-inflammatory drugs (NSAIDs) remain the most frequently prescribed drugs and common option for the treatment of inflammatory pain. However, they have the potential to cause serious complications, such as gastrointestinal (GI) lesions, bleeding and cardiovascular (CV) problems. NSAIDs exert their anti-inflammatory, analgesic and anti-pyretic actions by inhibiting the cyclooxygenases (COX)-1 and COX-2, key enzymes of the arachidonic acid (AA) cascade. However, not all the AA products or their receptors are pro-inflammatory. Therefore, given the multifaceted interactions of these lipid mediators where a single precursor can trigger multiple events with synergic or opposed function, it is easy to predict that any perturbation of this interplay will cause several unavoidable side effects. Today, we do not have a truly safe NSAID that minimizes GI damage and CV toxicity. One possibility to interfere with this intricate network, while trying to keep its fine balance, is to develop molecules affecting several targets. Different strategies have been proposed for a multitargeted intervention at different levels of the AA cascade, like inhibition of multiple upstream enzymes, such as COX, 5-lipoxygenase, or even soluble epoxide hydrolase and prostaglandin E synthase. Alternative strategies are more focused in the inhibition of targets downstream in the metabolic pathway, such as thromboxane synthase and/or blocking selective receptors. In this review we will briefly summarize the new strategies that have been proposed for a multitargeted pharmacological intervention on this metabolic cascade aimed at developing novel anti-inflammatory therapeutics.

Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology.

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.

Nonsteroidal Anti-Inflammatory Drugs: Exploiting Bivalent COXIB/ TP Antagonists for the Control of Cardiovascular Risk.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are some of the most widely prescribed or dispensed over the counter analgesics and antipyretics that act by inhibiting prostaglandins and thromboxane synthesis. After the identification of a second isoform of COX, the pharmaceutical research focused on developing COX-2- selective drugs (COXIBs) considered as second generation NSAIDs that would retain the anti-inflammatory and analgesic activities of traditional NSAID without blunting the gastrointestinal cytoprotection sustained by COX1-derived products such as PGE2. However, while several clinical trials confirmed a gastrointestinal safer profile of COXIBs vs unselective COX inhibitors, increasing evidence for potential cardiovascular risk associated with COXIBs rapidly emerged. Today, there are no really safe NSAIDs to be used in chronic pain and anti-inflammatory treatments, as an adequate therapy associated with a minimal gastrointestinal damage and cardiovascular toxicity is yet to be developed. OBJECTIVE: Here, we present evidences that combining the anti-aggregating and antiatherotrombotic activities of a thromboxane receptor antagonist with the antiinflammatory activity of a COXIB we could obtain a new multitarget drug providing protection against the harmful activities mediated by the COXIB component, yet exploiting its recognized therapeutic advantages as a gastrointestinal-safer anti-inflammatory drug. We also summarize recent progress achieved in this field of research and possible new strategies to obtain a new bivalent compound. CONCLUSION: This possible third-generation NSAID with a safer pharmacological profile, will have all the pharmacological characteristics for the long-term therapy of chronic disorders such as inflammatory diseases or selected forms of cancer.

The leukotriene receptor antagonist montelukast and its possible role in the cardiovascular field.

BACKGROUND: Cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are pro-inflammatory mediators of the 5-lipooxygenase (5-LO) pathway, that play an important role in bronchoconstriction, but can also enhance endothelial cell permeability and myocardial contractility, and are involved in many other inflammatory conditions. In the late 1990s, leukotriene receptor antagonists (LTRAs) were introduced in therapy for asthma and later on, approved for the relief of the symptoms of allergic rhinitis, chronic obstructive pulmonary disease, and urticaria. In addition, it has been shown that LTRAs may have a potential role in preventing atherosclerosis progression. PURPOSE: The aims of this short review are to delineate the potential cardiovascular protective role of a LTRA, montelukast, beyond its traditional use, and to foster the design of appropriate clinical trials to test this hypothesis. RESULTS AND CONCLUSIONS: What it is known about leukotriene receptor antagonists? •Leukotriene receptor antagonist, such as montelukast and zafirlukast, is used in asthma, COPD, and allergic rhinitis. • Montelukast is the most prescribed CysLT1 antagonist used in asthmatic patients. • Different in vivo animal studies have shown that leukotriene receptor antagonists can prevent the atherosclerosis progression, and have a protective role after cerebral ischemia. What we still need to know? • Today, there is a need for conducting clinical trials to assess the role of montelukast in reducing cardiovascular risk and to further understand the mechanism of action behind this effect.

The DRY motif and the four corners of the cubic ternary complex model.

Recent structural data on GPCRs using a variety of spectroscopic approaches suggest that GPCRs adopt a dynamic conformational landscape, with ligands stabilizing subsets of these states to activate one or more downstream signaling effectors. A key outstanding question posed by this emerging dynamic structural model of GPCRs is what states, active, inactive, or intermediate are captured by the numerous crystal structures of GPCRs complexed with a variety of agonists, partial agonists, and antagonists. In the early nineties the discovery of inverse agonists and constitutive activity led to the idea that the active receptor state (R⁎) is an intrinsic property of the receptor itself rather than of the RG complex, eventually leading to the formulation of the cubic ternary complex model (CTC). Here, by a careful analysis of a series of data obtained with a number of mutants of the highly conserved E/DRY motif, we show evidences for the existence of all the receptor states theorized by the CTC, four 'uncoupled (R, R⁎ and HR and HR⁎), and, consequently four 'coupled' (RG, R⁎G, HRG and HR⁎G). The E/DRY motif located at the cytosolic end of transmembrane helix III of Class A GPCRs has been widely studied and analyzed because it forms a network of interactions believed to lock receptors in the inactive conformation (R), and, thus, to play a key role in receptor activation. Our conclusions are supported by recent crystal and NMR spectra, as well as by results obtained with two prototypical GPCRs using a new FRET technology that de-couples G protein binding to the receptor from signal transduction. Thus, despite its complexity and limitations, we propose that the CTC is a useful framework to reconcile pharmacological, biochemical and structural data.

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPß homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.

Design and Characterization of Superpotent Bivalent Ligands Targeting Oxytocin Receptor Dimers via a Channel-Like Structure.

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.

In vitro pharmacological evaluation of multitarget agents for thromboxane prostanoid receptor antagonism and COX-2 inhibition.

PURPOSE: Patients with high cardiovascular risk due to ageing and/or comorbidity (diabetes, atherosclerosis) that require effective management of chronic pain may take advantage from new non-steroidal anti-inflammatory drugs (NSAIDs) that at clinical dosages may integrate the anti-inflammatory activity and reduced gastrointestinal side effects of selective cyclooxygenase-2 (COX-2) inhibitor (coxib) with a cardioprotective component involving antagonism of thromboxane A2 prostanoid (TP) receptor. METHODS: New compounds were obtained modulating the structure of the most potent coxib, lumiracoxib, to obtain novel multitarget NSAIDs endowed with balanced coxib and TP receptor antagonist properties. Antagonist activity at TP receptor (pA2) was evaluated for all compounds in human platelets and in an heterologous expression system by measuring prevention of aggregation and Gq-dependent production of intracellular inositol phosphate induced by the stable thromboxane A2 (TXA2) agonist U46619. COX-1 and COX-2 inhibitory activities were assessed in human washed platelets and lympho-monocytes suspension, respectively. COX selectivity was determined from dose-response curves by calculating a ratio (COX-2/COX-1) of IC50 values. RESULTS: The tetrazole derivative 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at TP receptor, preventing human platelet aggregation and intracellular signalling, with pA2 values statistically higher from that of lumiracoxib. Comparative data regarding COX-2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitor, compound 20 was somehow less potent and selective for COX-2. CONCLUSION: These results indicate that compounds 18 and 20 are two novel combined TP receptor antagonists and COX-2 inhibitors characterized by a fairly balanced COX-2 inhibitor activity and TP receptor antagonism and that they may represent a first optimization of the original structure to improve their multitarget activity.

A potential role of PUFAs and COXIBs in cancer chemoprevention.

Polyunsaturated fatty acids (PUFAs), particularly the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution processes are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.

Transcellular biosynthesis of eicosanoid lipid mediators.

The synthesis of oxygenated eicosanoids is the result of the coordinated action of several enzymatic activities, from phospholipase A2 that releases the polyunsaturated fatty acids from membrane phospholipids, to primary oxidative enzymes, such as cyclooxygenases and lipoxygenases, to isomerases, synthases and hydrolases that carry out the final synthesis of the biologically active metabolites. Cells possessing the entire enzymatic machinery have been studied as sources of bioactive eicosanoids, but early on evidence proved that biosynthetic intermediates, albeit unstable, could move from one cell type to another. The biosynthesis of bioactive compounds could therefore be the result of a coordinated effort by multiple cell types that has been named transcellular biosynthesis of the eicosanoids. In several cases cells not capable of carrying out the complete biosynthetic process, due to the lack of key enzymes, have been shown to efficiently contribute to the final production of prostaglandins, leukotrienes and lipoxins. We will review in vitro studies, complex functional models, and in vivo evidences of the transcellular biosynthesis of eicosanoids and the biological relevance of the metabolites resulting from this unique biosynthetic pathway. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Pharmacogenetics of the G protein-coupled receptors.

Pharmacogenetics investigates the influence of genetic variants on physiological phenotypes related to drug response and disease, while pharmacogenomics takes a genome-wide approach to advancing this knowledge. Both play an important role in identifying responders and nonresponders to medication, avoiding adverse drug reactions, and optimizing drug dose for the individual. G protein-coupled receptors (GPCRs) are the primary target of therapeutic drugs and have been the focus of these studies. With the advance of genomic technologies, there has been a substantial increase in the inventory of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms and insertion or deletions that have potential to alter GPCR expression of function. In vivo and in vitro studies have determined functional roles for many GPCR variants, but genetic association studies that define the physiological impact of the majority of these common variants are still limited. Despite the breadth of pharmacogenetic data available, GPCR variants have not been included in drug labeling and are only occasionally considered in optimizing clinical use of GPCR-targeted agents. In this chapter, pharmacogenetic and genomic studies on GPCR variants are reviewed with respect to a subset of GPCR systems, including the adrenergic, calcium sensing, cysteinyl leukotriene, cannabinoid CB1 and CB2 receptors, and the de-orphanized receptors such as GPR55. The nature of the disruption to receptor function is discussed with respect to regulation of gene expression, expression on the cell surface (affected by receptor trafficking, dimerization, desensitization/downregulation), or perturbation of receptor function (altered ligand binding, G protein coupling, constitutive activity). The large body of experimental data generated on structure and function relationships and receptor-ligand interactions are being harnessed for the in silico functional prediction of naturally occurring GPCR variants. We provide information on online resources dedicated to GPCRs and present applications of publically available computational tools for pharmacogenetic studies of GPCRs. As the breadth of GPCR pharmacogenomic data becomes clearer, the opportunity for routine assessment of GPCR variants to predict disease risk, drug response, and potential adverse drug effects will become possible.

Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.

The endogenous ligands for the LT, lipoxin (LX) and oxoeicosanoid receptors are bioactive products produced by the action of the lipoxygenase family of enzymes. The LT receptors BLT1 and BLT2 , are activated by LTB4 and the CysLT1 and CysLT2 receptors are activated by the cysteinyl-LTs, whereas oxoeicosanoids exert their action through the OXE receptor. In contrast to these pro-inflammatory mediators, LXA4 transduces responses associated with the resolution of inflammation through the receptor FPR2/ALX (ALX/FPR2). The aim of the present review is to give a state of the field on these receptors, with focus on recent important findings. For example, BLT1 receptor signalling in cancer and the dual role of the BLT2 receptor in pro- and anti-inflammatory actions have added more complexity to lipid mediator signalling. Furthermore, a cross-talk between the CysLT and P2Y receptor systems has been described, and also the presence of novel receptors for cysteinyl-LTs, such as GPR17 and GPR99. Finally, lipoxygenase metabolites derived from ω-3 essential polyunsaturated acids, the resolvins, activate the receptors GPR32 and ChemR23. In conclusion, the receptors for the lipoxygenase products make up a sophisticated and tightly controlled system of endogenous pro- and anti-inflammatory signalling in physiology and pathology.

Functional characterization of E. coli LptC: interaction with LPS and a synthetic ligand.

Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt­LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS­LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 µm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 µM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.

Rosuvastatin inhibits human airway smooth muscle cells mitogenic response to eicosanoid contractile agents.

BACKGROUND: The concept of permanent narrowing of the airways resulting from chronic inflammation and fibrosis is called remodeling and is a common feature of asthma and chronic obstructive pulmonary disease (COPD). The eicosanoid contractile agents thromboxane A(2) (TxA(2)) and cysteinyl-leukotriene D(4) (LTD(4)) are among the recognized mitogens for human airway smooth muscle (ASM) cells. Statins are known to possess anti-inflammatory and immunomodulatory properties that are independent on their cholesterol-lowering effects and may result in clinical lung benefits. Rosuvastatin is the last agent of the lipid-lowering drugs to be introduced and experimental evidence indicates that it possess favorable pleiotropic effects in the cardiovascular and nervous systems. Yet, no data is available in the literature regarding its effects on human airway remodeling. The present study was aimed at examining the effect of rosuvastatin and the involvement of prenylated proteins in the response of human ASM cells to serum, epidermal growth factor (EGF) and eicosanoid contractile mitogens that activate TxA(2) prostanoid and LTD(4) receptors. METHODS: Cell growth was assessed by nuclear incorporation of [(3)H]thymidine in human ASM cells serum-starved and then stimulated for 48 h in MEM plus 0.1% BSA containing mitogens in the absence and presence of modulators of the mevalonate and prenylation pathways. RESULTS: We found that rosuvastatin dose-dependently inhibited serum-, EGF-, the TxA(2) stable analog U46619-, and LTD(4)-induced human ASM cells growth. All these effects were prevented by pretreatment with mevalonate. Addition of the prenylation substrates farnesol and geranylgeraniol reversed the effect of rosuvastatin on EGF and U46619, respectively. Interestingly, only mevalonate showed restoration of cell growth following rosuvastatin treatment in LTD(4) and LTD(4) plus EGF treated cells, suggesting a possible involvement of both farnesylated and geranylgeranylated proteins in the cysteinyl-LT-induced cell growth. CONCLUSIONS: The hydrophilic statin rosuvastatin exerts direct effects on human ASM cells mitogenic response in vitro by inhibiting prenylation of signaling proteins, likely small G proteins. These findings are consistent with previous observed involvement of small GTPase signaling in EGF- and U46619-induced human airway proliferation and corroborate the recent interest in the potential clinical benefits of statins in asthma/COPD.